Low Concentrations of Caffeine and Its Analogs Extend the Lifespan of Caenorhabditis elegans by Modulating IGF-1-Like Pathway

Abstract

Caffeine has been reported to delay aging and protect aging-associated disorders in Caenorhabditis elegans. However, the effects of low concentration of caffeine and its analogs on lifespan are currently missing. Herein, we report that at much lower concentrations (as low as 10 μg/ml), caffeine extended the lifespan of C. elegans without affecting food intake and reproduction. The effect of caffeine was dependent on IGF-1-like pathway, although the insulin receptor homolog, daf-2 allele, e1371, was dispensable. Four caffeine analogs, 1-methylxanthine, 7-methylxanthine, 1,3-dimethylxanthine, and 1,7-dimethylxanthine, also extended lifespan, whereas 3-methylxanthine and 3,7-dimethylxanthine did not exhibit lifespan-extending activity.

Keywords: C. elegans; IGF-1 pathway; caffeine; daf-2; lifespan.

FIGURE 1

Low concentrations of caffeine increased C. elegans’ lifespan from the period of egg hatching. (A,B) Low concentration of caffeine increased C. elegans’ lifespan in a dose-dependent manner. (C,D) Caffeine treatment at various time points extended lifespan. (E) 50 μg/ml and 100 μg/ml caffeine did not reduce pumping rate, and 15 worms were used for each group. (F) 50 μg/ml and 100 μg/ml caffeine did not affect brood size, and 15 worms were used for each group. (G) 50 μg/ml and 100 μg/ml caffeine did not affect body length (head-to-tail). The log-rank (Mantel-Cox) test was used to analyze the differences. Error bars, values are expressed as mean ± SEM; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 2

Effects of caffeine on lifespan from the time of egg hatching of N2 wild-type (WT) and IGF-1 pathway mutant strains. (A) N2 wild-type and daf-2 (e1371) mutant strain; (B) N2 wild-type and daf-2 (e1370) mutant strain; (C) N2 wild-type and age-1 (hx546) mutant strain; (D) N2 wild-type and akt1 (ok525) mutant strain; (E) N2 wild-type and akt-2 (ok393) mutant strain; (F) N2 wild-type and daf-16 (mu86) mutant strain. The log-rank (Mantel-Cox) test was used to analyze the differences. Error bars, values are expressed as mean ± SEM; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 3

Acute caffeine (50 μg/ml) exposure translocates DAF-16 to the nucleus. The GFP localization status was characterized into three types defined as (a–c), as shown in the figure. The numbers of each type were counted and percentages were calculated. N = 176 and 197, respectively.

FIGURE 4

Caffeine modulated the AKT signaling pathway. (A) Caffeine increased longevity-related gene expression which acted downstream of AKT. (B) Caffeine prohibited expression and phosphorylation of AKT 1/2/3 (Thr308); (C–E) Quantification of expression and phosphorylation of AKT. One-way analysis of variance (ANOVA). Error bars, values are expressed as mean SEM; ^∗p < 0.05; ^∗∗p < 0.01.

FIGURE 5

Effects of caffeine analogs on C. elegans’ lifespan from the period of egg hatching. (A–H) Four caffeine analogs, 1-methyl xanthine, 7-methyl xanthine, 1,3-dimethyl xanthine, and 1,7-dimethyl xanthine, also extended worm lifespan. Xanthine, 1-methyl xanthine, 3-methyl xanthine, 7-methyl xanthine, and 3,7-dimethyl xanthine were dissolved in DMSO to a final concentration of 0.1%, therefore 0.1% DMSO served as control. The log-rank (Mantel-Cox) test was used to analyze differences. Error bars, values are expressed as mean ± SEM; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.